Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins

The mechanism of the chromophore maturation in members of the green fluorescent protein (GFP) family such as DsRed and other red fluorescent and chromoproteins was analyzed. The analysis indicates that the red chromophore results from a chemical transformation of the protonated form of the GFP-like chromophore, not from the anionic form, which appears to be a dead-end product. The data suggest a rational strategy to achieve the complete red chromophore maturation utilizing substitutions to favor the formation of the neutral phenol in GFP-like chromophore. Our approach to detect the neutral chromophore form expands the application of fluorescent timer proteins to faster promoter activities and more spectrally distinguishable fluorescent colors. Light sensitivity found in the DsRed neutral form, resulting in its instant transformation to the mature red chromophore, could be exploited to accelerate the fluorescence acquisition.     

Insight into the common mechanism of the chromophore formation in the red fluorescent proteins: the elusive blue intermediate revealed

Understanding the chromophore maturation process in fluorescent proteins is important for the design of proteins with improved properties. Here, we present the results of electronic structure calculations identifying the nature of a blue intermediate, a key species in the process of the red chromophore formation in DsRed, TagRFP, fluorescent timers, and PAmCherry. The chromophore of the blue intermediate has a structure in which the π-system of the imidazole ring is extended by the acylimine bond, which can be represented by the model N-[(5-hydroxy-1H-imidazole-2yl)methylidene]acetamide (HIMA) compound. Ab initio and QM/MM calculations of the isolated model and protein-bound (mTagBFP) chromophores identify the anionic form of HIMA as the only structure that has absorption that is consistent with the experiment and is stable in the protein binding pocket. The anion and zwitterion are the only protonation forms of HIMA whose absorption (421 and 414 nm, or 2.95 and 3.00 eV) matches the experimental spectrum of the blue form in DsRed (the absorption maximum is 408 nm or 3.04 eV) and mTagBFP (400 nm or 3.10 eV). The QM/MM optimization of the protein-bound anionic form results in a structure that is close to the X-ray one, whereas the zwitterionic chromophore is unstable in the protein binding pocket and undergoes prompt proton transfer. The computed excitation energy of the protein-bound anionic form of the mTagBFP-like chromophore (3.04 eV) agrees with the experimental absorption spectrum of the protein. The DsRed-like chromophore formation in red fluorescent proteins is revisited on the basis of ab initio results and verified by directed mutagenesis revealing a key role of the amino acid residue 70, which is the second after the chromophore tripeptide, in the formation process.     

Low-temperature chromophore isomerization reveals the photoswitching mechanism of the fluorescent protein Padron

Photoactivatable fluorescent proteins are essential players in nanoscopy approaches based on the super-localization of single molecules. The subclass of reversibly photoswitchable fluorescent proteins typically activate through isomerization of the chromophore coupled with a change in its protonation state. However, the interplay between these two events, the details of photoswitching pathways, and the role of protein dynamics remain incompletely understood. Here, by using a combination of structural and spectroscopic approaches, we discovered two fluorescent intermediate states along the on-switching pathway of the fluorescent protein Padron. The first intermediate can be populated at temperatures as low as 100 K and results from a remarkable trans-cis isomerization of the anionic chromophore taking place within a protein matrix essentially deprived of conformational flexibility. This intermediate evolves in the dark at cryotemperatures to a second structurally similar but spectroscopically distinct anionic intermediate. The final fluorescent state, which consists of a mixture of anionic and neutral chromophores in the cis configuration, is only reached above the glass transition temperature, suggesting that chromophore protonation involves solvent interactions mediated by pronounced dynamical breathing of the protein scaffold. The possibility of efficiently and reversibly photoactivating Padron at cryotemperatures will facilitate the development of advanced super-resolution imaging modalities such as cryonanoscopy.     

Theoretical studies on the isomerization mechanism of the ortho-green fluorescent protein chromophore

We present a systematic theoretical investigation on the overall ground state and excited-state isomerization reaction mechanism of ortho-green fluorescent protein chromophore (o-HBDI) using the density functional theory and the multireference methods. The calculated results and subsequent analysis suggest the possible isomerization mechanism for o-HBDI. By comparison with experimental observation and detailed analysis, it is concluded that as initiated by the excited-state intramolecular proton transfer reaction, the conical intersection between the ground state and the excited state along the C4-C5 single-bond rotational coordinate is responsible for the rapid deactivation of o-HBDI.     

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield and thereby brightness of a dim monomeric RFP (mRojoA, quantum yield = 0.02) by optimizing chromophore packing with aliphatic residues, which we hypothesized would reduce torsional motions causing non-radiative decay. Experimental characterization of the top 10 designed sequences yielded mSandy1 (λ_em = 609 nm, quantum yield = 0.26), a variant with equivalent brightness to mCherry, a widely used RFP. We next used directed evolution to further increase brightness, resulting in mSandy2 (λ_em = 606 nm, quantum yield = 0.35), the brightest Discosoma sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore p-hydroxybenzylidene moiety is sandwiched between the side chains of Leu63 and Ile197, a structural motif that has not previously been observed in RFPs, and confirms that aliphatic packing leads to chromophore rigidification. Our results demonstrate that computational protein design can be used to generate bright monomeric RFPs, which can serve as templates for the evolution of novel far-red fluorescent proteins.

We used computational design to increase quantum yield in a fluorescent protein by optimizing chromophore packing to reduce non-radiative decay, resulting in an >10-fold increase in quantum yield that was further improved by directed evolution.     

Electronic excitations of green fluorescent proteins: modeling solvatochromatic shifts of red fluorescent protein chromophore model compound in aqueous solutions

While green fluorescent proteins (GFPs) have been widely used as tools in biochemistry, cell biology, and molecular genetics, novel red fluorescent proteins (RFPs) with red fluorescence emission have also been identified, as complements to the existing GFP technology. The unusual spectrophotometric and fluorescence properties of GFPs and RFPs are controlled by the protonation states and possibly cis/trans isomerization within their chromophores. In this work, we have investigated the electronic structures, liquid structures, and solvent shifts of the possible neutral and anionic protonated states and the cis/trans isomerization of a RFP chromophore model compound HBMPI in aqueous solutions. The calculations reproduced the experimental absorption solvatochromatic shifts of dilute HBMPI in water under neutral and anionic conditions. Unlike the GFP chromophore, the RFP chromophore model compound HBMPI in basic solution can only adopt a conformation where the C=C bond between the bridge group and the imidazolinone ring and the C-C bond between the imidazolinone and ethylene groups exist in cis and trans conformations, respectively. Moreover, the solvent-solute hydrogen-bonding interactions are found to contribute significantly to the total solvent shifts of pi-pi* excitations of aqueous HBMPI solutions, signifying the importance of protein environment in the determination of the conformation of the chromophores in red fluorescent proteins.     

Single oligomer spectra probe chromophore nanoenvironments of tetrameric fluorescent proteins

When analyzing the emission of a large number of individual chromophores embedded in a matrix, the spread of the observed parameters is a characteristic property for the particular chromophore-matrix system. To quantitatively assess the influence of the matrix on the single molecule emission parameters, it is imperative to have a system with a well-defined chromophore nanoenvironment and the possibility to alter these surroundings in a precisely controlled way. Such a system is available in the form of the visible fluorescent proteins, where the chromophore nanoenvironment is defined by the specific protein sequence. We analyze the influence of the chromophore embedding within this defined protein environment on the distribution of the emission maximum wavelength for a number of variants of the fluorescent protein DsRed, and show that this parameter is characteristic of the chromophore-protein matrix combination and largely independent of experimental conditions. We observe that the chemical changes in the vicinity of the chromophore of different variants do not account for the different distributions of emission maximum positions but that the flexibility of the chromophore surrounding has a dominant role in determining the distribution. We find, surprisingly, that the more rigid the chromophore surrounding, the broader the distribution of observed maximum positions. We hypothesize that, after a thermally induced reorientation in the chromophore surrounding, a more flexible system can easily return to its energetic minimum position by fast reorientation, while in more rigid systems the return to the energetic minimum occurs in a stepwise fashion, leading to the broader distribution observed.     

Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed

Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.     

Synthesis and trans-cis isomerization of azobenzene dendrimers incorporating 1,2-isopropylidenefuranose rings

Azobenzene dendrimer 2 was synthesized from a known dendritic azo-tetracarboxylic acid and a dendritic amine incorporating 1,2-isopropylidenefuranose rings, and its trans-cis isomerization was studied by UV-vis absorption spectroscopy.     

trans-cis Photoisomerization of a photoactive yellow protein model chromophore in crystalline phase

We have studied the photoinduced trans/cis isomerization of the protonated form of p-hydroxycinnamic thiophenyl ester, a model chromophore of the photoactive yellow protein (PYP), in crystalline phase, by both fluorescence and infrared spectroscopies. The conversion from trans to cis configuration is revealed by a shift of the fluorescence peak and by inspection of the infrared maker bands. The crystal packing apparently stabilizes the cis photoproduct, suggesting different environmental effects from the solvent molecules for this model chromophore in liquid solutions or from the amino acid residues for the PYP chromophore.     

Radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge-transfer states

We use CASSCF and MRPT2 calculations to characterize the bridge photoisomerization pathways of a model red fluorescent protein (RFP) chromophore model. RFPs are homologues of the green fluorescent protein (GFP). The RFP chromophore differs from the GFP chromophore via the addition of an N-acylimine substitution to a common hydroxybenzylidene-imidazolinone (HBI) motif. We examine the substituent effects on the manifold of twisted intramolecular charge-transfer (TICT) states which mediates radiationless decay via bridge isomerization in fluorescent protein chromophore anions. We find that the substitution destabilizes states associated with isomerization about the imidazolinone-bridge bond and stabilizes states associated with phenoxy-bridge bond isomerization. We discuss the results in the context of chromophore conformation and quantum yield trends in the RFP subfamily, as well as recent studies on synthetic models where the acylimine has been replaced with an olefin.     

Peptide bond formation catalyzed by α-chymotrypsin in ionic liquids

α-Chymotrypsin catalyzed peptide bond formation was studied in ionic liquids using the synthesis of a protected fragment of Leu-enkephalin, ZTyrGlyGlyOEt, as model reaction. MOEMIM·PF₆ was found to be the most favorable solvent among the six different 1-alkyl-3-methylimidazolium hexafluorophosphates and tetrafluoroborates ionic liquids screened. With MOEMIM·PF₆ as reaction media, several di- or tripeptide derivatives were successfully prepared in 68-75% isolated yields.     

INDO-SCF and CI calculations on the trans-cis isomerization of azomethane in the ground state and in excited states

INDO-SCF calculations followed by CI calculations with inclusion of multiply, excited configurations were carried out to obtain potential energy curves for isomerization in the ground state and in some low-lying excited states of azomethane. The SCF wavefunctions are analyzed with the aid of newly defined bond characters providing a connection between the chemical concepts of bonds, lone-pairs, etc. and molecular orbital theory. Two different pathways for isomerization are considered and by comparison of the calculated results with experimental data it is concluded that this reaction proceeds in the ^1,3 (nπ*) states via rotation of both methyl groups around the NN double bond.     

Deactivation mechanism of the green fluorescent chromophore

We report time-resolved fluorescence data for the anion of p-hydroxybenzylidene dimethylimidazolinone (p-HBDI), a model chromophore of the green fluorescence protein, in viscous glycerol-water mixtures over a range of temperatures, T. The markedly nonexponential decay of the excited electronic state is interpreted with the aid of an inhomogeneous model possessing a Gaussian coordinate-dependent sink term. A nonlinear least-squares fitting routine enables us to achieve quantitative fits by adjusting a single activation parameter, which is found to depend linearly on 1/T. We derive an analytic expression for the absolute quantum yield, which is compared with the integrated steady-state fluorescence spectra. The microscopic origins of the model are discussed in terms of two-dimensional dynamics, coupling the phenyl-ring rotation to a swinging mode that brings this flexible molecule to the proximity of a conical intersection on its multidimensional potential energy surface.     

Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611

An important class of red fluorescent proteins (RFPs) feature a 2-iminomethyl-5-(4-hydroxybenzylidene)imidazolinone chromophore. Among these proteins, eqFP611 has the chromophore in a coplanar trans orientation, whereas the cis isomer is preferred by other RFPs such as DsRed and its variants. In the photoactivatable protein asFP595, the chromophore can even be switched from the nonfluorescent trans to the fluorescent cis state by light. By using X-ray crystallography, we have determined the structure of dimeric eqFP611 at high resolution (up to 1.1 A). In the far-red emitting eqFP611 variant d2RFP630, which carries an additional Asn143Ser mutation, the chromophore resides predominantly (approximately 80%) in the cis isomeric state, and in RFP639, which has Asn143Ser and Ser158Cys mutations, the chromophore is found completely in the cis form. The pronounced red shift of excitation and emission maxima of RFP639 can thus unambiguously be assigned to trans-cis isomerization of the chromophore. Among RFPs, eqFP611 is thus unique because its chromophore is highly fluorescent in both the cis and trans isomeric forms.     

Electronic excitations of green fluorescent proteins: protonation states of chromophore model compound in solutions

Green fluorescent proteins (GFPs) are widely used as tools in biochemistry, cell biology, and molecular genetics due to their unusual optical spectroscopic characteristics. The spectrophotometric and fluorescence properties of GFPs are controlled by the protonation states and possibly cis-trans isomerization of the chromophore (p-hydroxybenzylideneimidazolinone). In this work, we have investigated electronic structures, liquid structures, and solvent shifts of the three possible protonated states (neutral, anionic, and zwitterionic) and their cis-trans isomerization of a model compound 4'-hydroxybenzylidene-2-methyl-imidazolin-5-one-3-acetate (HBMIA) in aqueous solutions. Our calculated results suggest that HBMIA could adopt both cis and trans conformations in a solution, and it exists in three different protonation states depending on the pH conditions. The absorption spectrum observed in neutral solution is thus assigned to the electronic excitations within the neutral form and the cis isomer of the zwitterionic form, while the absorption band at 425 nm in the basic solution is due to the excitations within the anionic form and the trans isomer of the zwitterionic form. Some technical problems related to the computation of electronic excitations within the HBMIA at the anionic state are also discussed.     

Isomerization mechanism of the HcRed fluorescent protein chromophore

To understand how the protein achieves fluorescence, the isomerization mechanism of the HcRed chromophore is studied both under vacuum and in the solvated red fluorescent protein. Quantum mechanical (QM) and quantum mechanical/molecular mechanical (QM/MM) methods are applied both for the ground and the first excited state. The photoinduced processes in the chromophore mainly involve torsions around the imidazolinone-bridge bond (τ) and the phenoxy-bridge bond (φ). Under vacuum, the isomerization of the cis-trans chromophore essentially proceeds by τ twisting, while the radiationless decay requires φ torsion. By contrast, the isomerization of the cis-trans chromophore in HcRed occurs via simultaneous τ and φ twisting. The protein environment significantly reduces the barrier of this hula twist motion compared with vacuum. The excited-state isomerization barrier via the φ rotation of the cis-coplanar conformer in HcRed is computed to be significantly higher than that of the trans-non-coplanar conformer. This is consistent with the experimental observation that the cis-coplanar-conformation of the chromophore is related to the fluorescent properties of HcRed, while the trans-non-planar conformation is weakly fluorescent or non-fluorescent. Our study shows how the protein modifies the isomerization mechanism, notably by interactions involving the nearby residue Ile197, which keeps the chromophore coplanar and blocks the twisting motion that leads to photoinduced radiationless decay.     

Excited-state structure determination of the green fluorescent protein chromophore

Ultrafast polarization-sensitive infrared (IR) spectroscopy of the C=O stretching mode of the chromophore of the green fluorescent protein reveals a near complete twisting around the ethylenic bridge between the phenolate and imidazolidinone groups upon electronic excitation, hinting at a decisive role of this motion in the efficient internal conversion process.     

Slow exchange in the chromophore of a green fluorescent protein variant

Green fluorescent protein and its mutants have become valuable tools in molecular biology. They also provide systems rich in photophysical and photochemical phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. Here, we report the (19)F nuclear magnetic resonance (NMR) studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow molecular motions in these proteins. The concerted use of dynamic NMR and (19)F relaxation measurements, supported by temperature, concentration- and folding-dependent experiments provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. (19)F NMR relaxation and line shape analysis indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodynamic analysis revealed a difference in enthalpy (Delta)H(0) = (18.2 +/- 3.8) kJ/mol and entropy T(Delta)S(0) = (19.6 +/- 1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estimated to be approximately 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("atomic mutations") like H --> F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.